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Abstract

Reptiles have achieved highly diverse morphological and physiological traits that
allow them to exploit various ecological niches and resources. Morphology of the temporal
region of the reptilian skull is highly diverse and historically it has been treated
as an important character for classifying reptiles and has helped us understand the
ecology and physiology of each species. However, the developmental mechanism that
generates diversity of reptilian skull morphology is poorly understood. We reveal
a potential developmental basis that generates morphological diversity in the temporal
region of the reptilian skull by performing a comparative analysis of gene expression
in the embryos of reptile species with different skull morphology. By investigating
genes known to regulate early osteoblast development, we find dorsoventrally broadened
unique expression of the early osteoblast marker, Runx2, in the temporal region of the head of turtle embryos that do not form temporal fenestrae.
We also observe that Msx2 is also uniquely expressed in the mesenchymal cells distributed at the temporal region
of the head of turtle embryos. Furthermore, through comparison of gene expression
pattern in the embryos of turtle, crocodile, and snake species, we find a possible
correlation between the spatial patterns of Runx2 and Msx2 expression in cranial mesenchymal cells and skull morphology of each reptilian lineage.
Regulatory modifications of Runx2 and Msx2 expression in osteogenic mesenchymal precursor cells are likely involved in generating
morphological diversity in the temporal region of the reptilian skull.

Keywords:

Reptiles; Skull; Morphology; Development; Osteogenesis; Heterotopy

Introduction

Amniotes (Amniota) consist of two large groups of tetrapod vertebrates, Synapsida
and Reptilia, that diverged from one another over 300 million years ago (Ma) (Carroll,
1988;Modesto & Anderson,, Modesto & Anderson, Modesto & Anderson,2004, Benton, 2005). The synapsids are represented today by mammals while reptiles by extant turtles,
tuatara, lizards, snakes, crocodiles, birds, and their extinct relatives, including
dinosaurs and pterosaurs. Over time, reptiles have evolved highly diverse morphological
and physiological traits that allow them to exploit various ecological niches and
resources on the land, in water, and in the air.

Morphology of the skull of reptiles, especially the temporal region is highly diverse
(Figure 1). This morphological diversity observed in the temporal region is broadly categorized
into three architectural patterns. The anapsid condition of the skull is observed
in basal amniotes such as Scutosaurus and Captorhinus, and in turtles. In these animals, the temporal region of the skull is completely
roofed by bones, without temporal openings (fenestrae). The synapsid condition of
the skull is recognized in ancestral lineages of extant mammals and is characterized
by the presence of a temporal fenestra at lower position of either side of the skull.
The diapsid condition of the skull is seen in "non-turtle" extant reptiles and in
their extinct relatives. In these animals, two temporal fenestrae exist on either
side of the skull. In reptiles with fully diapsid skulls, the upper temporal fenestra
is dorsally bordered by the parietal bone, anteriorly by the postorbital bone, and
posteriorly by the squamosal bone. The lower temporal fenestra is dorsally surrounded
by both the postorbital and squamosal bones, and ventrally by both the jugal and quadratojugal
bones. These temporal fenestrae are thought to have evolved to allow space for accommodating
the enlarged jaw-closing muscles that enable powerful biting, or to minimize stresses
exerted by the contraction of jaw muscles on the skull, or to reduce the weight of
the skull itself (Frazzetta, 1968, Carroll, 1982, Rieppel, 1993a, Benton, 2005). During the course of diapsid evolution skull morphology has been rearranged repeatedly,
resulting in a variety of modified patterns (Rieppel, 1993a;Rieppel & Gronowski,, Rieppel & Gronowski, Rieppel & Gronowski,1981;Müller,, Müller, Müller,2003, Moazen et al., 2009). Among extant reptiles, fully diapsid skull is only seen in tuatara and crocodiles.
Because the lower temporal bar that encloses the lower temporal fenestra ventrally
is regarded to be lost once in the common ancestor of lepidosaurs (lizards, snakes,
tuatara) and archosaurs (crocodiles and birds) (Rieppel, 1993a;Müller,, Müller, Müller,2003), these reptilian lineages possibly acquired the lower temporal bar secondarily (Müller,
2003, Moazen et al., 2009). Both snakes and birds have lost the upper temporal bar so that their temporal region
is free from any bony frames (Pough et al., 2005).

Figure 1.Simplified phylogeny of the Reptilia highlighting diversity of their skull morphology. Paleontological evidence suggests that all reptiles, including extant lizards, snakes,
tuatara, crocodiles, birds, and turtles, were derived from ancestor whose temporal
region was completely roofed by bone. Earliest diapsid reptiles such as Petrolacosaurus (Araeoscelidia) acquired two temporal openings (fenestrae) on either side of the
skull (red vertical bar). Recent molecular phylogenies indicate that turtles (Testudines)
were derived from diapsid ancestor, which would require secondary closure of temporal
fenestrae (green vertical bar). The bone surrounding anteroventral border of the upper
temporal fenestra and anterodorsal border of the lower temporal fenestra in diapsids:
the postorbital was colored in pink. The bone surrounding posteroventral border of
the upper temporal fenestra and posterodorsal border of the lower temporal fenestra
in diapsids: the squamosal was colored in blue. The bone surrounding the anteroventral
margin of the lower temporal fenestra in diapsids: the jugal was colored in yellow.
Lizards do not have the lower temporal bar. Both upper and lower temporal bars are
absent in snakes and birds. Other extinct diapsid lineages such as Ichthyosauria and
Sauropterygia were not included in phylogeny for simplicity.

Although skull morphology has been regarded as an important character in classification
of reptiles and in understanding the ecological and physiological aspects of each
reptilian species, the developmental mechanism underlying diversification of reptilian
skull morphology is poorly understood (Rieppel, 1993a, Evans, 2008). As a consequence, a general genetic and developmental model of reptile skull diversity
does not yet exist. In this paper, we test the hypothesis that changes of skeletal
gene expression patterns cause diversification of reptilian skull morphology through
comparative analyses of gene expression in the embryos of representative reptilian
species and reveal a potential developmental basis underlying reptilian skull evolution.
First, we describe the pattern of early phases of cranial morphogenesis in a crocodile
species with both upper and lower temporal bars surrounding temporal fenestrae, using
molecular markers specific for musculoskeletal tissue precursors. Then, we compare
these data with cranial morphogenesis in a turtle species. We found a broader expression
of the early osteogenic genes, Runx2 and Msx2 in the mesenchymal cells at the temporal region of turtle embryos, compared to that
in crocodile embryos. Finally, to obtain a broader picture of reptilian skull morphogenesis,
we examined expression patterns of Runx2 and Msx2 in cranial morphogenesis of a snake species without temporal bars on the skull and
compared with the patterns in crocodile and turtle embryos. Our findings suggest that
there is a possible correlation between the expression patterns of Runx2 and Msx2 and the architectural pattern seen in the temporal region of the reptilian skull.

Differential expression of early osteoblast marker, Runx2, in the head of crocodile and turtle embryos

Osteogenic mesenchymal precursor cells that express Runx2 are first detected in the temporal region of crocodile embryos at stage 14 (Additional
file 1) and an almost identical pattern of Runx2 expression was observed in a subsequent embryonic stage (stage 15; Figure 2A). These Runx2-positive cells were localized at the domain dorsal to the oral cavity where the ventral
part of the braincase and future palatine and pterygoid bones develop, as well as
in a limited domain dorsolateral to the orbit where the future dorsal projection of
the postorbital bone forms (Figure 2B). We also detected a population of Runx2-positive cells at the domain ventrolateral to the orbit where future jugal bone and
ventral projection of postorbital bone are formed. At this stage, the precursor of
the jugal and ventral projection of the postorbital were dorsoventrally continued
as a layer of cells but it was thin mediolaterally, especially at the middle part.
In the posterior part of the head, we observe a population of Runx2-positive mesenchymal cells that later differentiate into the main body of the postorbital
bone (Figures 2E and 2H). In these stages of crocodile embryogenesis, jaw muscle precursors that were derived
from cranial mesoderm migrated to the first pharyngeal arch and expressed MyoD was clearly detected at the central domain of the jaw primordia (Figures 2C and 2F; Additional file 1). Expression of Sox9 was detected at cartilage precursors that later differentiate into quadrate and Meckel's
cartilages at the domain ventral to jaw muscle precursor, as well as in chondrocytes
that form the future braincase (Figures 2D and 2G). Expression of Scx was detected in tendon precursor cells that are distributed within the primordia
of the jaw muscles and in the connective tissue within the eye muscles (Additional
file 1). Expression of Six2 was somewhat broader than that of other markers, expressed in mesenchymal cells surrounding
the eyes, cartilaginous precursors of the braincase, quadrate, and Meckel's, as well
as in the mesenchyme at the interface between muscle precursors and the skeletal tissues
to which the muscles attach and in a population of the mesenchyme that dorsally surrounds
the brain (Additional file 1).

Figure 2.Expression of musculoskeletal tissue marker genes in the head of crocodile embryos
at stage 15. (A) Lateral view of the head of a crocodile embryo at stage 15. (B-D) Frontal sections prepared around the plane indicated by the red line in (A). (E-G) Frontal sections prepared around the plane indicated by the blue line in (A). (H) Frontal sections prepared around the plane indicated by the green line in (A). (B, E, and H)Runx2-positive mesenchymal cells are observed at the location where future dermatocranial
elements are developed. (C and F) Expression of MyoD is detected at the cranial muscular tissues. (D and G) Cartilaginous tissues, including the braincase, quadrate, and Meckel's, are clearly
labeled by Sox9 probe. The red outlined domains in (B, D, E, and G) indicate the location of the pseudotemporal muscle (ptm) deduced from adjacent sections
where muscular tissues are labeled by MyoD probe. Scale bar in (A) is 1 mm. Scale bars in (B-H) are 0.5 mm.

Additional file 1.Expression of musculoskeletal tissue marker genes in the head of crocodile embryos
at stage 14. (A) Lateral view of the embryo. (B-D, and F) Frontal sections prepared around the
plane indicated by the blue line in (A). (E) Frontal section prepared around the plane
indicated by the red line in (A). (B) Expression of Runx2 is faintly detected at the mesenchymal cells above and below the eye, as well as
in the mesenchyme distributed medial to the precursor of quadrate cartilage and in
the mesenchyme surrounding the braincase (arrows). (C) Cranial muscular tissues are
clearly labeled by MyoD probe. (D) Cartilaginous tissues, including the braincase and the quadrate (q), are
labeled by Sox9 probe. (E) Scx is expressed in tendon precursor cells in close proximity of MyoD-positive jaw and eye muscle anlagen (arrowheads). (F) Six2 is expressed mainly in the mesenchyme around Sox9-positive cartilage precursors, including the quadrate and the braincase, as well
as in the mesenchyme around MyoD-positive cranial muscle anlagen. The red outlined domains indicate the location of
the anlagen of the jaw muscle complex. Scale bar in (A) is 1 mm. Scale bars in (B-F)
are 0.5 mm.

In crocodile embryos at stage 17 where none of the dermatocranial elements were positive
for Alizarin red in previous studies (Rieppel, 1993b;Vickaryous & Hall,, Vickaryous & Hall, Vickaryous & Hall,2008) (Figure 3A), we could detect Runx2 expression in the cell populations that were localized to the area where the future
dermatocranium differentiates (Figure 3B). Although the domain where Runx2-positive cells were populated was almost identical to that in previous stages, the
boundary of each precursor of the dermatocranial elements became clearer. Although
differentiation of the parietal bone is delayed compared to other dermatocranial elements
as described previously (Rieppel 1993b;Vickaryous & Hall,, Vickaryous & Hall, Vickaryous & Hall,2008), a pair of precursors of the parietal were recognized as Runx2-positive cell aggregation at the domain dorsolateral to the cartilaginous braincase
(Figures 3B and 3E). We could detect Runx2-negative cell populations between the nascent parietal precursor and the morphologically
more developed postorbital precursor and also between precursors of the postorbital
and quadratojugal located lateral to Sox9-positive quadrate cartilage (Figure 3E). In this stage, MyoD was expressed in differentiated jaw and eye muscles (Figures 3C and 3F) and Sox9 was expressed in differentiated chondrocranium and splanchnocranium components, including
the braincase, quadrate, and Meckel's (Figures 3D and 3G). Expression of Scx was detected in tendinous tissues accompanying MyoD-positive muscles as in previous stages (Figure 3H). Expression of Six2 was detected in the mesenchyme localized around the jaw articulation between quadrate
and Meckel's, as well as in adjacent mesenchyme of the braincase, postorbital bone,
and jaw muscles (Figure 3I).

Figure 3.Expression of musculoskeletal tissue marker genes in the head of crocodile embryos
at stage 17. (A) Lateral view of the head of a crocodile embryo at stage 17. (B-D) Frontal sections prepared around the plane indicated by the red line in (A). (E-I) Frontal sections prepared around the plane indicated by the blue line in (A). (B and E) Expression of Runx2 is more concentrated to the precursors of dermatocranial elements, compared to the
previous stages. (C, D, F, and G) Cranial muscular and cartilaginous tissues are clearly labeled by MyoD and Sox9 probes, respectively. (H and I) Expression domains of Scx and Six2 are indicated by arrowheads. The former is expressed in tendinous tissues accompanying
cranial muscles and the latter is expressed mainly in connective tissue cells associated
with cartilages of the jaw and the braincase. The red outlined domains in (B, D, E, G, H, and I) indicate the location of the pseudotemporal muscle deduced from adjacent sections
where muscular tissues are labeled by MyoD probe. Green line in (A) indicates the plane where sections given in Figure 7D, E and F were prepared. Scale bar in (A) is 1 mm. Scale bars in (B-I) are 0.5 mm.

Next, we examined cranial morphogenesis of turtles that have an anapsid skull, using
the same method to identify the distribution pattern of precursors of each tissue
that constitutes the cranial musculoskeletal system. In turtle embryos at stage 14
(Additional file 2) that correspond to crocodile embryos at stage 14 in external morphology, and in
turtle embryos at stage 15 (Figure 4A) that are comparable to crocodile embryos at stage 15, we observed almost identical
patterns of expression for each gene. We detected MyoD expression specifically in the primordia of jaw adductor and eye muscles (Figures 4C and 4H; Additional file 2) and Sox9 expression in precursor cells of the braincase, quadrate, and Meckel's cartilages
(Figures 4D and 4I; Additional file 2) as in stage-matched crocodile embryos. Expression of Scx was first detected in a layer of mesenchymal cells that was located at the periphery
of the jaw adductor and eye muscle precursors in stage 15 turtle embryos (Figure 4E). Expression of Six2 was observed in the mesenchyme surrounding the eye and adjacent mesenchyme of the
braincase and jaw cartilages, as well as in some mesenchymal cells within jaw muscle
precursors (Figure 4F; see Additional file 2), as in stage-matched crocodile embryos. Interestingly, we observed expression of
the early osteoblast marker, Runx2, in a broader domain at the temporal region of the head of turtle embryos, compared
to that in stage-matched crocodile embryos. In stage14 turtle embryos, Runx2 expression was detected in a population of cells medial to the precursor of the jaw
adductor muscles and the mesenchyme localized at the domain dorsolateral and ventrolateral
to the orbit (Additional file 2). The domain of Runx2 expression became further expanded in the head of turtle embryos at stage 15. A thick
layer of the mesenchymal cells that express Runx2 completely covered the brain and the precursor of jaw adductor muscle laterally (Figures 4B and 4G).

Figure 4.Expression of musculoskeletal tissue marker genes in the head of turtle embryos at
stage 15. (A) Lateral view of the head of a turtle embryo at stage 15. (B-F) Frontal sections prepared around the plane indicated by the red line in (A). (G-I) Frontal sections prepared around the plane indicated by the blue line in (A). (B, G)Runx2-positive mesenchymal cells (arrows) are broadly distributed at lateral portion of
the head, from the top of the head to the ventral margin of the first pharyngeal arch.
(C and H) Cranial muscular tissues are clearly labeled by MyoD probe. (D and I) Cartilaginous tissues, including the braincase and quadrate, are clearly labeled
by Sox9 probe. (E)Scx is expressed in tendon primordia accompanying cranial muscles (arrowheads). (F)Six2 is expressed mainly in connective tissue cells associated with the braincase cartilage
and jaw adductor muscle (arrowheads). Note that the anlage of jaw adductor muscle
(red outlined domain) is covered by a thick layer of Runx2-positive mesenchyme laterally. Scale bar in (A) is 1 mm. Scale bars in (B-I) are 0.5 mm.

Additional file 2.Expression of musculoskeletal tissue marker genes in the head of turtle embryos at
stage 14. (A) Lateral view of the embryo. (B-E) Frontal sections prepared around the plane
indicated by the red line in (A). (B) Runx2-positive mesenchymal cells are distributed above and below the eye, as well as in
the domain medial to the anlagen of jaw adductor muscle (black arrows). (C) Cranial
muscular tissues are clearly labeled by MyoD probe. (D) Cartilaginous tissues, including the braincase and quadrate, are labeled
by Sox9 probe. (E) Six2 is expressed mainly in the mesenchyme around Sox9-positive cartilage precursors and MyoD-positive cranial muscle anlagen. Scale bar in (A) is 1 mm. Scale bars in (B-E) are
0.5 mm.

In turtle embryos at stage 17 (Figure 5A) that correspond to crocodile embryos at stage 17 in overall morphology, we observed
MyoD expression in differentiating cranial muscles, including external adductor muscles
(Figure 5C) and Sox9 expression in the cartilaginous tissues that constitute the braincase, quadrate,
and Meckel's (Figure 5D). Scx was specifically expressed in tendinous tissues at the periphery of jaw adductor
muscles, as well as in the precursor of the bodenaponeurosis (central tendon of external
adductor) just appeared within the jaw adductor muscular tissue (Figure 5E). The expression domain of Six2 was broader in the temporal region of the head compared to that of Scx, diffusively expressed in the mesenchymal cells surrounding jaw adductor muscles,
braincase, and jaw cartilages (Figure 5F). A thick layer of Runx2-positive mesenchymal cells that surrounds the braincase and jaw adductor muscle laterally
was observed (Figure 5B). Runx2-expressing mesenchyme was also distributed around the quadrate cartilage and the
ventral part of the braincase.

Figure 5.Expression of musculoskeletal tissue marker genes in the head of turtle embryos at
stage 17. (A) Lateral view of the head of a turtle embryo at stage 17. (B-F) Frontal sections prepared around the plane indicated by the blue line in (A). (B)Runx2-positive mesenchymal cells (arrows) are broadly distributed at the lateral portion
of the head, from the top of the head to the ventral margin of the first pharyngeal
arch derivative. (C) Cranial muscular tissues are clearly labeled by MyoD probe. (D) Cartilaginous tissues, including the braincase and quadrate, are clearly labeled
by Sox9 probe. (E)Scx is expressed in tendinous tissues at the periphery of external adductor muscle (ame)
(arrowheads) and in the bodenaponeurosis (boap. central tendon of jaw adductor muscle).
(F)Six2 is expressed mainly in connective tissue cells associated with cartilages of the
braincase and quadrate, as well as in connective tissue cells within jaw muscles (arrowheads).
Note that the external adductor muscle (red outlined domain) is covered by a thick
layer of Runx2-positive mesenchyme laterally. Scale bar in (A) is 1 mm. Scale bars in (B-F) are 0.5 mm.

Expression of potential upstream osteogenic regulatory genes in the head of crocodile
and turtle embryos

Through comparative analysis of expression patterns of tissue-specific marker genes,
we noticed a difference in the spatial pattern of expression of the early osteoblast
marker, Runx2 in the head of crocodile and turtle embryos. To reveal potential mechanisms that
account for such differential distribution of osteogenic mesenchymal precursor cells
between two reptilian lineages with or without temporal fenestrae, we next examined
expression patterns of some candidate genes that are known to regulate cranial osteogenesis.
In the present study, we focused on Bmp4, Msx1, and Msx2. Bmp4 is a signaling molecule and plays a key role in the Bmp signaling pathway.
Because exogenous Bmp4 increases tissue volume in calvarial bone tissue culture, this
protein is considered to be involved in calvarial bone growth (Kim et al., 1998, Rice et al., 2003). Both Msx1 and Msx2 are members of the muscle segment homeobox (msh) gene family
of transcription factors and both loss-of- and gain-of-function analyses of these
genes suggest their essential roles in vertebrate cranial osteogenesis (Satokata &
Maas, 1994, Satokata et al., 2000).

In the present analysis, we found that Bmp4 and Msx1 showed almost identical expression patterns through cranial osteogenesis in crocodile
and turtle embryos. In crocodile embryos we examined (through stage 14 to stage 17),
Bmp4 was strongly expressed in the epithelium of cochlear canal, the mesenchyme surrounding
the eye, the mesenchyme distributed in the medial part of jaw primordia, the precursors
of the palatine bones, and a population of mesenchymal cells that covered the brain
dorsally (Figure 6A; Figures 7A and 7D). In turtle embryos we examined (through stage 14 to stage 17), Bmp4 was expressed in a spatially limited domain: the epitthelium of cochlear canal, the
mesenchyme dorsolateral and ventrolateral to the eye and a limited population of the
mesenchyme in close proximity of the jaw articulation (Figure 6G; Figure 7G). We observed Msx1 expression in the epithelium of the cochlear canal, the mesenchyme that occupies
the domain close to the jaw articulation and lateral to the quadrate and Meckel's
cartilages, and a thin layer of mesenchymal cells that covers the brain dorsally in
crocodile embryos examined (Figure 6B; Figures 7B and 7E). In turtle embryos, Msx1 was expressed in the epithelium of the cochlear canal, the mesenchyme distributed
around the jaw articulation and lateral to quadrate and Meckel's cartilages, as well
as in the mesenchyme that populates the domain dorsal to the eye (Figure 6H; Figure 7H).

Figure 6.Expression of Bmp4, Msx1, and Msx2 in crocodile and turtle embryos at stage 15. (A-D) Frontal sections prepared around the plane indicated by the red line in Figure 2A. (E, F) Frontal sections prepared around the plane indicated by the blue line in Figure 2A. (G-J) Frontal sections prepared around the plane indicated by the red line in Figure 4A. (K, L) Frontal sections prepared around the plane indicated by the blue line in Figure 4A. (A and G) In both crocodile and turtle embryos, expression of Bmp4 is detected at the mesenchyme distributed in medial part of the jaw primordia (arrows).
(B and H) Expression of Msx1 is detected at the mesenchyme that occupies the domain close to jaw articulation
and lateral to quadrate and Meckel's cartilages (arrows). (C and E) In crocodile embryos, Msx2 is expressed in a thin layer of mesenchymal cells surrounding dorsal aspect of the
brain and in a population of the mesenchyme that occupies the domain between ventrolateral
part of quadrate cartilage and surface epidermis (arrows). (I and K) In turtle embryos, Msx2 is expressed in mesenchymal cells that populate lateral aspect of the head (arrows).
In contrast to the condition in crocodile embryos, the ventral edge of Msx2-expressing mesenchymal layer is terminated ventral to the eye in turtle embryos and
these cells cover MyoD-expressing jaw adductor muscle precursor (J and L) laterally. The red outlined domains in (A-C, and E) indicate the location of the anlagen of the pseudotemporal muscle deduced from adjacent
sections where muscular tissues are labeled by MyoD probe. The red outlined domains in (G-I, and K) indicate the location of the anlagen of jaw adductor muscle. Scale bars are 0.5 mm.

Figure 7.Expression of Bmp4, Msx1, and Msx2 in crocodile and turtle embryos at stage 17. (A-C) Frontal sections prepared around the plane indicated by the blue line in Figure 3A. (D-F) Sections prepared around the plane indicated as the green line in Figure 3A. (G-I) Sections prepared around the plane indicated as the red line in Figure 5A. (J) Section prepared around the plane indicated as the blue line in Figure 5A. (A, D, and G) In both crocodile and turtle embryos, expression of Bmp4 is detected in the epithelium of the cochlear canal, the mesenchyme surrounding the
eye, and the mesenchyme distributed in medial part of the jaw (arrows). (B, E, and H) Expression of Msx1 is detected at the mesenchymal cells that later differentiates into quadratojugal
bone (qj) and in a thin layer of mesenchymal cells that covers brain dorsally (arrows
at the top of E and H), as well as in the epithelium of the cochlear canal. (C and F) In crocodile embryos, expression of Msx2 is detected at a population of mesenchymal cells in close proximity of postorbital
and quadratojugal bones, as well as in a layer of the mesenchyme surrounding the brain
dorsally where future a pair of parietal bones are developed. (I and J) In turtle embryos, Msx2 was expressed in a thick layer of mesenchymal cells that populate lateral aspect
of the head (arrows). The Msx2-positive mesenchymal cells cover the external adductor muscle precursors (red outlined
domains in G-J) laterally. The red outlined domains in (A-F) indicate the location of the pseudotemporal muscle deduced from adjacent sections
where muscular tissues are labeled by MyoD probe. The blue outlined domains in (A-J) indicate the location of quadrate cartilage deduced from adjacent sections where
cartilaginous tissues are labeled by Sox9 probe. Scale bars are 0.5 mm.

In contrast to Bmp4 and Msx1, we detected differential expression patterns of Msx2 in the head of crocodile and turtle embryos. In crocodile embryos at stage 14 and
15, Msx2 was expressed in a thin layer of mesenchymal cells surrounding the dorsal aspect
of the brain (Figures 6C and 6E). In the posterior part of the head, the ventral edge of this Msx2-expressing cell population is located dorsal to the eye. In these crocodilian embryos,
Msx2 expression was also observed in a population of the mesenchyme that occupied the
domain between the ventrolateral part of quadrate cartilage and surface epidermis
(Figures 6C and 6E). These mesenchymal cells expressed Msx1 as well (Figure 6B) and appeared to differentiate into the quadratojugal bone later. In crocodile embryos
at stage 17, specific expression of Msx2 was detected at a population of mesenchymal cells in close proximity of Runx2-expressing precursors of postorbital and quadratojugal bones, as well as in a thin
layer of the mesenchyme surrounding the brain dorsally where future parietal bones
were developed (Figures 7C and 7F). We observed that the space adjacent to Msx2-positive precursors of these dermatocranial elements was filled with Msx2-negative mesenchymal cells. Interestingly, we observed broader expression of Msx2 in turtle embryos, compared to that in stage-matched crocodile embryos. In turtle
embryos examined, Msx2 was expressed in mesenchymal cells that populate lateral aspect of the head of embryos
(Figures 6I and 6K; Figures 7I and 7J). The ventral edge of the Msx2-expressing mesenchymal layer was terminated ventral to the eye and these cells covered
MyoD-expressing external adductor muscle laterally. Showing its dorsoventrally broadened
expression pattern, the domain of Msx2 expression largely overlapped with that of Runx2 in turtle embryos (Figures 4B and 4G; Figure 5B).

Expression of Runx2 and Msx2 in the head of snake embryos

To verify a correlation between the expression patterns of Runx2 and Msx2 and reptilian skull morphology, we finally examined expression patterns of these
genes, as well as of marker genes for muscular and cartilaginous tissues, in cranial
morphogenesis of a snake species where their temporal fenestrae are not encircled
by the temporal bars. In snake embryos at stage 26 (Figure 8A) that morphologically correspond to crocodile and turtle embryos at stage 14 or
15, MyoD was expressed in the primordia of the first arch muscles (Figures 8C and 8G). Sox9 was strongly expressed in the precursors of quadrate and Meckel's cartilages and
the base of the braincase, as well as in a layer of mesenchyme surrounding the brain
laterally (Figures 8D and 8H). In these snake embryos, early osteoblast marker, Runx2 was expressed in the mesenchyme that occupied the space medial to the Sox9-positive quadrate precursor and in a limited population of mesenchymal cells ventral
to the orbit (Figure 8B). We also detected Runx2 expression in a layer of mesenchymal cells that surround the brain laterally. In
the posterior temporal region, Runx2 was only faintly expressed in the adjacent mesenchyme of the Sox9-positive quadrate precursor (Figure 8F). Expression of Msx2 was detected in the mesenchyme medial to the precursor of the quadrate (Figure 8E). Its expression domain was spatially overlapped with the domain where Runx2-positive cells were distributed, but the former was narrower. We also detected Msx2 expression in a mesenchymal layer that surrounded the brain dorsally (Figures 8E and 8I). In snake embryos at stage 29 (Figure 8J), which morphologically correspond to crocodile and turtle embryos at stage 17,
well-differentiated cranial muscular and cartilaginous tissues were specifically labeled
by expression of MyoD (Figures 8L and 8P) and Sox9 (Figures 8M and 8Q), respectively. We observed spatially overlapped expression of Runx2 and Msx2 in these embryos: both genes were expressed in the precursors of palatine and pterygoid
bones medial to Sox9-positive jaw cartilages and in mesenchymal cells accompanying jaw cartilages, as
well as in a layer of loose mesenchyme that later forms a precursor of parietal bones
that cover the dorsal part of the brain (Figures 8K, 8N, 8O, and 8R). However, only Runx2 was expressed in the mesenchyme that populated the domain ventral to the orbit, which
possibly corresponds to precursors of the maxilla bones of the upper jaw. No Runx2 and/or Msx2 expressing osteogenic mesenchymal precursor cells populated the domain lateral to
jaw adductor muscles. In more developed snake embryos (at stage 31; Figure 8S), both Runx2 and Msx2 were specifically expressed in the precursors of the dermatocranial elements, including
the parietal that encases the brain dorsally (Figures 8T and 8W). As in previous stages, expression of the former was more expanded. The lateral
aspect of the external adductor muscles was never covered by the skeletal tissues
that express Runx2 and/or Msx2. The results on expression domains of each gene analyzed in crocodile, turtle, and
snake embryos are summarized in Table 1.

Figure 8.Expression of the genes that regulate the development of cranial musculoskeletal tissues
in snake embryos. (A) Lateral view of the head of a snake embryo at stage 26. (B-E) Frontal sections prepared around the plane indicated by the red line in (A). (F-I) Frontal sections around the blue line in (A). (J) The head of a snake embryo at stage 29. (K-N) Frontal sections around the red line in (J). (O-R) Frontal sections around the blue line in (J). (S) The head of a snake embryo at stage 31. (T-W) Frontal sections around the red line in (S). (B, F, K, O, and T) Expression of Runx2. (C, G, L, P, and U) Expression of MyoD. (D, H, M, Q, and V) Expression of Sox9. (E, I, N, R, and W) Expression of Msx2. In snake embryos, expression of Runx2 and Msx2 are detected in the precursor cells of dermatocranial elements (arrows), as in crocodile
and turtle embryos. However, these mesenchymal cells are not seen in the temporal
region lateral to the jaw adductor muscles. Instead, these cells are distributed in
the vicinity of the brain, laterally covering it, and differentiate into the parietal
bone in older embryos. Open arrowheads in (F, K, N, O, R, T, and W) indicate the mesenchyme around Sox9-positive jaw cartilages, where the expression of Runx2 and Msx2 is detected. The red outlined domains in (O, Q, R, T, V, and W) indicate the location of the external adductor muscle deduced from adjacent sections
where muscular tissues are labeled by MyoD probe. Scale bars in (A), (J), and (S) are 1 mm. Scale bars in other pictures are 0.5 mm.

Table 1.Expression domains of the genes in the head of crocodile, turtle, and snake embryos

Discussion

Potential developmental basis of anapsid skull in turtles

Skull morphology, especially the osteological configuration of the temporal region,
has historically been treated as the most important character in the classification
of major lineages of reptiles. Based on their anapsid skull, turtles have been regarded
as a sole descendent of stem reptiles (Williston, 1917, Gregory, 1946, Romer, 1968, Gaffney, 1980;Reisz & Laurin,, Reisz & Laurin, Reisz & Laurin,1991, Lee, 1993;Laurin & Reisz, 1995, Lee, 1996 1997, Reisz, 1997, Lee, 2001) despite the contrary argument that turtles were derived from an ancestor with a
diapsid skull (Lakjer, 1926, Goodrich, 1930). Recent phylogenetic studies where the interrelationships of both extant and extinct
reptiles were surveyed through comprehensive analysis of multiple osteological traits
concluded that turtles were closely related to lepidosaurian diapsids (Rieppel & deBraga,
1996;deBraga & Rieppel , deBraga & Rieppel deBraga & Rieppel 1997Rieppel 2000, Hill, 2005, Li et al., 2008). Furthermore, results of molecular phylogenetic studies have strongly suggested
diapsid affinity of turtles (Hedges & Poling, 1999;Kumazawa & Nishida,, Kumazawa & Nishida, Kumazawa & Nishida,1999, Iwabe et al., 2005, Hugall et al., 2007, Shedlock et al., 2007Shen et al. 2011, Tzika et al., 2011, Chiari et al., 2012, Crawford et al., 2012, Fong et al., 2012, Lyson et al., 2012, Wang et al., 2013). If turtles were derived from a diapsid ancestor, then the anapsid skull of turtles
evolved independently from that of ancestral lineages of reptiles by secondary closure
of the temporal fenestrae. However, although the phylogenetic position of turtles
within amniotes still remains inconclusive (Lyson et al., 2010 2013, Kuratani et al., 2011), there has been no study in which the process of development of their anapsid skull
is described with molecular markers for labeling precursor cells of the dermatocranium.
In the present study, we examined early cranial morphogenesis of representative reptilian
species through comparative analysis of gene expression patterns and found unique
expression patterns of Runx2 and Msx2 in turtle embryos that are not observed in crocodile and snake embryos.

Runx2 is widely known as a transcription factor that plays a fundamental role in osteoblast
differentiation in vertebrate embryos (Ducy et al., 1997, Komori et al., 1997, Mundlos et al., 1997, Nakashima et al., 2002, Ishii et al., 2003, Dobreva et al., 2006, Kerney et al., 2010) and its transcript has been used as a molecular marker for preosteoblasts or osteoblast
progenitors (Ducy et al., 1997, Karsenty, 2001, Bobola et al., 2003, Ishii et al., 2003, Abzhanov et al., 2007, Han et al., 2007;Tokita & Schneider , Tokita & Schneider Tokita & Schneider 2009). In turtle embryos where mineralization of dermal bones of the skull has not yet
occurred, mesenchymal cells that express Runx2 were broadly distributed in the lateral domain of the head, from the top of the head
to the ventral margin of the jaw (Figure 9). This means that turtle embryos have wider distribution of the cells that have a
potential to differentiate into bones in the temporal region of the head, compared
to other lineages of reptiles. The reduction of the amount of Runx2 mRNA causes developmental defects in calvarial bones, called cleidocranial dysplasia,
in mouse embryos (Lou et al., 2009). In contrast, early onset of Runx2 expression that eventually results in an increase of the amount of its mRNA in the
cranial mesenchyme accelerates the timing of mineralization of cranial dermal bones
in mouse embryos and brings about craniosynostosis characterized by overgrowth of
bones (Maeno et al., 2011). Similar result has been obtained from experiments using avian embryos (Merrill
et al., 2008). We speculate that heterotopy in the preosteoblast distribution observed in early
stage turtle embryos may lead to the increase of the amount of Runx2 expression that results in the increase of the level of ossification in the temporal
region of their skull and this would be a primary factor to build the anapsid skull
where the temporal region is completely covered with bone.

Figure 9.Potential developmental basis that generates morphological diversity in the temporal
region of the reptilian skull. All extant reptilian lineages are considered to be derived from ancestor with diapsid
skull. In crocodiles that have both upper and lower temporal bars like the stem Diapsida
(e.g., Petrolacosaurus), osteogenic mesenchymal precursor cells which express Runx2 and/or Msx2 are distributed at the domain where future temporal bars are formed in the head of
early stage embryo (top of the middle column). Through ontogeny (black arrow), these
osteoblast precursors may differentiate into the dermatocranial elements including
upper and lower temporal bars (bottom of the middle column). Between these bony bars,
both upper and lower temporal fenestrae are clearly recognized. In turtles (left white
arrow), distribution of osteogenic mesenchymal precursor cells is broadened in a dorsal-ventral
direction, filling the whole lateral portion of the head of early embryos (top of
the left column). Through ontogeny, these osteoblast precursors may differentiate
into the dermatocranial elements roofing the temporal region of the head (bottom of
the left column). In snakes that have modified diapsid skull where temporal bars are
absent, osteogenic mesenchymal precursor cells do not fill lateral domain of the head
of embryo. Rather, these cells are mainly distributed in the vicinity of the brain
(top of the right column). Through ontogeny, these osteoblast precursors may differentiate
into the dermatocranial elements accompanying the braincase, without forming bony
temporal bars (bottom of the right column). A condensed mesenchymal layer that differentiates
into the braincase in later stages is highlighted by dotted line in the head of embryos.

In this study, we focused on several candidate molecules that potentially regulate
Runx2 expression and examined their expression patterns in reptilian embryos. Bmp4 is known
to be involved in osteogenesis of vertebrates where it regulates expression of other
osteogenic regulatory genes, including Msx1, Msx2, and Runx2 (Marazzi et al., 1997, Kim et al., 1998, Hollnagel et al., 1999, Tribulo et al., 2003, Zhang et al., 2003, Brugger et al., 2004). Msx1 is a transcription factor known to regulate growth and patterning of calvarial
bones in mouse embryos (Satokata & Maas, 1994, Han et al., 2007, Roybal et al., 2010). Although, as previously reported in mouse embryos (Rice et al., 2003, Han et al., 2007), both Bmp4 and Msx1 are expressed in limited populations of cranial mesenchyme in embryos of crocodiles
and turtles, we could not detect any substantial differences in their expression domains
between the two species. On the other hand, we observed spatially different expression
patterns of Msx2 in the head of embryos of all reptilian species we examined. Expression of Msx2 was detected in cranial mesenchyme and dermal bone precursors as reported in mouse
embryos (Jabs et al., 1993, Ishii et al., 2003, Rice et al., 2003, Han et al., 2007, Roybal et al., 2010). Furthermore, its expression spatially overlapped with that of Runx2 in reptilian embryos, as in mouse embryos (Ishii et al., 2003, Rice et al., 2003, Han et al., 2007). In turtle embryos, expression domain of Msx2 in the mesenchyme distributed in the temporal region of the head was broad in a dorsal-ventral
direction, showing similar pattern with Runx2 in the mesenchyme. A mutation in the homeobox of Msx2 gene causes craniosynostosis in human and mouse (Jabs et al., 1993, Liu et al., 1999). Similarly, overexpression of Msx2 promotes osteogenesis (Cheng et al., 2003, Ichida et al., 2004) and causes overgrowth of dermal bones of the skull by increasing the number of proliferative
osteoblasts (Dodig et al., 1999, Liu et al., 1999). In contrast, loss-of-function of Msx2 results in defects of skull ossification in mammals (Satokata et al., 2000, Wilkie et al., 2000, Ishii et al., 2003, Antonopoulou et al., 2004, Han et al., 2007). Furthermore, Msx2 is known to positively regulate downstream Runx2 expression (Ishii et al., 2003, Han et al., 2007, Watanabe et al., 2008). Considering the evidence provided by previous studies, regulatory changes in Msx2 expression in turtle embryos may influence expression patterns of downstream Runx2, which regulate osteoblast differentiation. Dorsoventrally broadened distribution
of osteogenic mesenchymal precursor cells in the temporal region of the head owing
to the regulatory alteration of these osteogenic genes may allow this reptilian lineage
to reacquire the anapsid skull. Although the precise mechanism underlying regulatory
change of Msx2 expression in the head of turtle embryos has not been identified, recent findings
that early stage arrest of Msx2 expression in neural crest-derived odontoblasts may account for the absence of teeth
in turtles (Tokita et al., 2012) supports the hypothesis that this transcription factor may play a pivotal role in
the development of their unique cranial morphology.

The development of the dermatocranium occurs in multiple steps (Ishii et al., 2003). The first phase includes the genesis, migration, and initial specification of osteogenic
mesenchymal precursor cells. The second phase consists of the differentiation of the
mesenchyme into osteoblasts. And the last phase includes deposition of osteogenic
extracellular matrix around the osteoblasts and mineralization of the matrix. The
dermatocranium of vertebrates is formed from cranial mesenchyme derived from two distinct
embryonic sources: neural crest and mesoderm (Jiang et al., 2002;Gross & Hanken , Gross & Hanken Gross & Hanken 2005;Noden & Trainor,, Noden & Trainor, Noden & Trainor,2005). Unfortunately, fate mapping studies of each dermatocranial element as performed
in avian and mammalian embryos (Le Lièvre, 1978, Noden, 1978 1983, Couly et al., 1993;Köntges & Lumsden,, Köntges & Lumsden, Köntges & Lumsden,1996, Jiang et al., 2002) have not been done in non-avian reptiles. Interestingly, the pattern of migration
and distribution of cranial neural crest cells from which some cranial dermal bones
should form is almost identical in early stage embryos of crocodiles and turtles (Meier
& Packard, 1984;Hou & Takeuchi,, Hou & Takeuchi, Hou & Takeuchi,1994;Kundrát,, Kundrát, Kundrát,2008). Such data may support that differentiation or maturation processes of osteogenic
mesenchyme are more responsible for producing diversity of reptilian skull morphology.
We speculate that the developmental program, which determines cranial mesenchymal
populations where early-phase osteogenic transcription factors Msx2 and Runx2 are
activated, may be important in the patterning of reptilian skull morphology.

There exists substantial diversity in the skull morphology within turtles and most
living turtle species do not have fully anapsid skulls and instead possess varying
degrees of dorsal and/or ventral emargination on their skull (Jones et al., 2012, Werneburg, 2012a). In the present study, we could not sample and analyze the embryos of turtle species
with fully anapsid skull, such as marine turtles (Kuratani, 1999, Jones et al., 2012), alligator snapping turtle () (Sheil, Macrochelys temminckii2005), and big-headed turtle (Platysternon megacephalum), owing to difficulty in the access to the materials. Instead, we analyzed the embryos
of a soft-shelled turtle species with highly emarginated skull. In fact, soft-shelled
turtles have only a narrow bar of bone across the temporal region lateral to the external
adductor muscles due to large scale emargination from the dorsal and ventral margins
of the cheek (Ogushi, 1911, Sheil, 2003). In normal development of soft-shelled turtles, the postorbital bone does not grow
in a posterior direction significantly, keeping its relatively small size within the
dermatocranium (Sheil, 2003;Sánchez-Villagra et al.,, Sánchez-Villagra et al., Sánchez-Villagra et al.,2009). Therefore, the small postorbital bone of soft-shelled turtles does not largely
contribute to the formation of a bony roof at the temporal region of the skull.

It is interesting that we observed dorsoventrally broadened distribution of the mesenchymal
cells that express Runx2 at the temporal region of the embryos of a soft-shelled turtle species with highly
emarginated skull. Dermal bone development occurs through a multi-step molecular pathway
regulated by different transcription factors (Zhang, 2010). As an initial step, Runx2 is required for the differentiation of mesenchymal cells into preosteoblasts. In
subsequent stage where these preosteoblasts differentiate into mature osteoblasts,
Osx, a downstream gene of Runx2, is necessary (Nakashima et al., 2002, Nishio et al., 2006). Furthermore, in the later stages where the osteoblasts produce osteogenic extracellular
matrix and the mineralization of these extracellular matrix is occurred, many additional
molecules such as bone sialoprotein, osteopontin, and osteocalcin are involved (Zhang,
2010). We speculate that in soft-shelled turtles only a limited population of cells within
Runx2-positive preosteoblasts distributed in the temporal region of the head is allowed
to differentiate into mature osteoblasts and eventually osteocytes through regulation
of expression of down stream genes (e.g. Osx), to form a pair of relatively small postorbital bones. Although the regulatory mechanism
of Osx expression in osteogenic mesenchyme is not fully understood, both Runx2-dependent
and -independent pathways have been suggested (Lee et al., 2003;Celil & Campbell,, Celil & Campbell, Celil & Campbell,2005, Maehata et al., 2006, Xing et al., 2007, Zhang, 2010). Histological analysis reveals that late stage soft-shelled turtle embryos have
a layer of (non-muscular) fibrous connective tissue lateral to the external adductor
muscles (Additional file 3). Judging from its position, the connective tissue layer appears to be derived from
Runx2-positive preosteoblasts and have a potential to ossify themselves as other connective
tissues represented by tendons and ligaments (Okawa et al., 1998;Tokita et al., 2007). Interestingly, similar type of connective tissue layer is absent in the temporal
region of crocodile and snake embryos (Additional file 3). Those histological observations support the above hypothesis that later processes
of cranial osteogenesis may largely contribute to the construction of the main body
of each dermatocranial element from the osteogenic mesenchymal progenitor pool.

Additional file 3.A layer of fibrous connective tissue lateral to the external adductor muscle is found
in late stage soft-shelled turtle embryo. (A) Lateral view of the head of a turtle embryo at stage 22. (B, C) Frontal sections
of the head prepared in the planes indicated in (A). Note a clear layer of fibrous
connective tissue lateral to the external adductor muscle (red arrowheads). (D) Lateral
view of the head of a crocodile embryo at stage 20. (E, F) Frontal sections of the
head prepared in the planes indicated in (D). (G) Lateral view of the head of a snake
embryo at stage 31. (H, I) Frontal sections of the head prepared in the planes indicated
in (G). A layer of fibrous connective tissue is not seen in the domain lateral to
the external adductor muscle in crocodile and snake embryos. Rather, in crocodile
and snake embryos, the domain is occupied by mesenchymal cells in low density or by
acellular cavities. Scale bars are 1 mm.

The dorsoventrally broadened distribution of preosteoblasts observed in turtle embryos
might be a developmental synapomorphy re-acquired by the common ancestor of turtles.
In the course of chelonian evolution, each chelonian lineage may develop the temporal
dermal bones (e.g. postorbital, parietal, jugal) with various sizes and shapes, through
regulatory changes of the osteogenic down stream molecules. Future studies should
investigate expression pattern of Runx2 and Msx2 in the head of embryos of turtle species with fully anapsid skull, as well as expression
pattern of downstream genes that regulate differentiation of mature osteoblasts and
osteocytes in turtle embryos, to verify a correlation between the gene expression
pattern and their skull morphology.

The frame-like skulls possessed by diapsid reptiles evolved in response to functional
forces (Rieppel, 1993a, Moazen et al., 2009, Herrel et al., 2007, Curtis et al., 2011) and several studies have suggested heterochrony as a driving force for producing
this morphological diversity (Rieppel, 1993a, Whiteside, 1986, Irish, 1989). The ancestral lineage of diapsid reptiles possessed upper and lower temporal bars
that encircle temporal fenestrae (Müller, 2003, Moazen et al., 2009). The lower temporal bar that encloses lower temporal fenestra ventrally was probably
lost once in the common ancestor of lepidosaurs and archosaurs, possibly as the outcome
of paedomorphosis: incomplete ossification of a quadrato-maxillary ligament between
jugal and quadratojugal bones (Rieppel, 1993a;Müller,, Müller, Müller,2003). If this is true, the lower temporal bar that possibly results from peramorphosis
(hypermorphosis): complete ossification of a quadrato-maxillary ligament was independently
re-acquired in the lineages of tuatara and crocodiles, as well as in several extinct
reptilian lineages (Rieppel, 1993a;Müller,, Müller, Müller,2003). Furthermore, disappearance of upper temporal bar, which is regarded as an extreme
condition of reduction of the dermatocranium in reptiles, may have independently evolved
in the skull of geckos (Gekkonidae), miniaturized fossorial lizards (e.g., Typhlosaurus, Dibamus), amphisbaenian, and snakes, as the outcome of paedomorphosis represented by the
retardation of ossification (Rieppel, 1993a, Irish, 1989;Cundall & Irish,, Cundall & Irish, Cundall & Irish,2008). In the present study, we revealed a possible correlation between distribution pattern
of Runx2 and/or Msx2-expressing osteogenic mesenchymal precursor cells and the skull morphology of each
reptilian lineage (Figure 9). In early stage crocodile embryos, we observed focal distribution of osteogenic
mesenchyme around the domain where future temporal bars are formed. In early stage
snake embryos, osteogenic mesenchymal cells were primarily found adjacent to the primordium
of the braincase and the spatial pattern presaged the absence of bony temporal bars
in the temporal region of adult animal.

Conclusions

Regulatory modifications of Runx2 and Msx2 expression in osteogenic mesenchymal precursor cells are likely involved in generating
morphological diversity in the temporal region of the reptilian skull, including secondary
closure of the temporal fenestrae in turtles. Our findings demonstrate that not only
heterochrony in ossification of the dermatocranium that has been traditionally regarded
as the major factor producing diversity of reptilian cranial morphology but also heterotopy
in distribution of the osteogenic precursor cells may play a fundamental role in this
process and it should be further investigated in future studies of reptilian cranial
development and evolution.

Materials and methods

Sample collection and staging of embryos

Fertilized eggs of Chinese soft-shelled turtle, Pelodiscus sinensis, were purchased commercially from a local breeder in Japan. Fertilized eggs of Siamese
crocodile, Crocodylus siamensis, were provided by a local breeder in Thailand. Fertilized eggs of corn snake, Pantherophis guttatus, were obtained by the first author after mating several pairs of the reproductively
mature adults in the laboratory. Staging of P. sinensis embryos was performed after Tokita and Kuratani (2001). Because there is no embryonic staging system for C. siamensis at present, we used the system for Alligator mississippiensis embryos (Ferguson, 1985) where each stage was determined based on external morphology of the embryos, for
staging of this species. Staging of P. guttatus (Zehr, embryos was performed on the basis of staging table of Thamnophis sirtalis1962). Interspecific comparisons of gene expression pattern were performed in the embryos
that are comparable to each other in terms of overall external morphology. Because
snake embryos are limbless, we mainly employed external features of the head of the
embryos as primary criteria for determining the stages for comparison. All animal
experiments were approved by the University of Tsukuba Committee for Animal Care (No.10-034).

Molecular cloning

Total RNA was extracted from embryos using ISOGEN reagent (NIPPON GENE CO., LTD).

RT–PCR was performed to amplify fragments of P. sinensis Runx2, Six2 and C. siamensis Bmp4, Msx2, MyoD, Runx2, Scleraxis (Scx), Six2, Sox9 and P. guttatus Msx2, Runx2, Sox9 messenger RNA. Primer sequences used for isolation of the fragments of these genes
are available upon request. Because Bmp4, Msx1, Msx2, MyoD, Scx, Sox9 of Pelodiscus and MyoD of Pantherophis were already sequenced and sequence data were deposited in the database by other
researchers, we isolated the orthologous fragments by RT–PCR with primers constructed
by referring to the reported sequence data. The fragments were isolated using the
pGEM T-easy vector systems (Promega) or TOPO® TA cloning kit (Invitrogen) and sequenced
using an ABI 3130 sequencer (Applied Biosystems). To identify the orthologous genes
of the isolated fragments, comparable sequence data were surveyed using a BLAST search,
and phylogenetic trees with neighbor joining method were constructed after sequence
alignment using the CLUSTALX software. All new DNA sequence data were deposited in
the DDBJ database (AB811933-AB811944).

Gene expression analysis

Embryos were fixed in 4% PFA, dehydrated using an methanol series, placed in xylene,
embedded in paraffin, and sliced with a microtome. Serial sections were hybridized
with digoxigenin-labeled RNA riboprobes as described in Neubüser et al. (1995) with slight modifications. To identify the expression domain of Msx1 in crocodile tissues, chicken Msx1antisense riboprobe was hybridized. Generally, hetero-specific RNA probes easily hybridize
among reptilian lineages (Harris et al., 2006, Tokita et al., 2012). In this study, we only analyzed reptilian embryos at the ontogenetic stages where
early cranial osteogenesis occurs. To confirm the expression pattern of each gene
in the cranial tissues, two to five individuals representing each embryonic stage
were sampled for analysis. The consistency of the gene expression patterns among all
individual embryos at the same stage was confirmed. Multiple sections representing
several longitudinal (anterior-posterior) planes prepared from the same individual
were hybridized with the probes and the sections prepared at corresponding longitudinal
planes were compared between different individuals. Corresponding longitudinal planes
between different reptilian species were determined based on overall histological
configuration of the head of the embryos. For visualization of each cranial tissue
and interspecific comparison of general histology of the head, Miligan's Trichrome
staining was performed following standard protocols. To identify each anatomical structure
in cranial musculoskeletal tissues of the embryos, we took the results of other's
researches into account: (Schumacher, 1973, Rieppel, 1993b;Vickaryous & Hall,, Vickaryous & Hall, Vickaryous & Hall,2008;Bona & Desojo,, Bona & Desojo, Bona & Desojo,2011) for crocodile, (Schumacher, 1973, Rieppel, 1990, Rieppel, 1993c;Sánchez-Villagra et al.,, Sánchez-Villagra et al., Sánchez-Villagra et al.,2009, Werneburg, 2012a 2012b) for turtle, and (Kamal et al., 1970, Haas, 1973, Zaher, 1994;Buchtová et al.,, Buchtová et al., Buchtová et al.,2007) for snake.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

MT conceived and designed the study. MT, WC, and JS contributed to collection and
preparation of biological materials. MT performed experiments and analyses and wrote
the paper. All authors read, discussed and approved the final manuscript.

Acknowledgments

We appreciate Sriracha Crocodile Farm & Product Co., LTD. and the company staffs,
especially Nussara Thongprasert in collection of fertilized eggs of Crocodiles. We
also thank Manasaree Klomtun, Punnapa Pinweha, and Ekawit Threenet for their kind
help in collection of Crocodile eggs. MT thanks Hiroshi Wada who allowed the use of
facilities for experiments and analyses, Hiroki Ono who kindly gifted chick Msx1 probe, Matthew Brandley and Richard Schneider for critical reading and editing of
a draft of the manuscript, and Johannes Müller for helpful comments to early version
of the manuscript. This study was partially supported by Grants-in-Aid from the Ministry
of Education, Culture, Sports, Science and Technology of Japan to M.T. (22770077).

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